Zanthoxylum armatum peroxidase immobilized enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering materials, and particularly relates to zanthoxylum armatum peroxidase immobilized enzyme as well as a preparation method and application thereof. According to the invention, zanthoxylum armatum peroxidase is immobilized on a magnetic carrier to prepare the zanthoxylum armatum peroxidase immobilized enzyme with high activity, high stability and high cyclicity. The toxicity of a phenol solution treated by the immobilized enzyme to normal cells of a human body is remarkably weakened, the phenol solution is applied to a phenol wastewater bioremediation agent, development of zanthoxylum armatum peroxidase in degradation of phenol wastewater is promoted, a new solution is provided for comprehensive utilization and development of zanthoxylum armatum and remediation of phenol wastewater, and the method has a very good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering materials, and in particular relates to an immobilized Zanthoxylum bungeanum peroxidase enzyme and a preparation method and application thereof. Background Art

[0002] Zanthoxylum armatum DC is the fruit of the plant of the genus Zanthoxylum L. of the family Rutaceae. It is also known as "green pepper" in the southwestern part of Sichuan. Due to its advantages such as easy cultivation, fast growth, high yield, strong fragrance, pure numbing taste, and high oil content, the planting area in Yunnan, Guizhou, Sichuan and other places has continued to rise in recent years, and the output has surged, falling into the dilemma of overcapacity. At present, it is mainly used as primary food in the market, and there has been no major breakthrough in the fields of medicine and health care, high-end food, and chemical industry, and the comprehensive utilization rate of products is low. Therefore, the development of high-value-added innovative products using green pepper as raw materials has become the first choice to combat the problem of overcapacity.

[0003] Previous studies have found that Zanthoxylum bungeanum contains about 0.16% peroxidase, and compared with commercial horseradish peroxidase, Zanthoxylum bungeanum peroxidase (ZADTP) has better pH and temperature stability and a higher phenol removal rate, making it an ideal raw material for the development of plant enzyme-based phenol-containing wastewater remediation agents. However, free enzymes are easily affected by harsh environments and are difficult to recycle and reuse, which severely limits the practical application of Zanthoxylum bungeanum peroxidase.

[0004] Immobilized enzyme technology is a bioengineering technology developed in the 1960s. It is an important means to make biological enzymes widely and effectively used. Enzyme immobilization is a technology that uses solid materials to bind or restrict enzymes to a certain area, perform their unique catalytic reactions, and can be recovered and reused. Compared with free enzymes, immobilized enzymes not only maintain their high efficiency, specificity and mild enzyme catalytic reaction characteristics, but also overcome the shortcomings of free enzymes, showing advantages such as high storage stability, easy separation and recovery, repeated use, continuous and controllable operation, and simple process.

[0005] At present, immobilization technology has achieved many important results, giving full play to the huge potential of immobilized enzymes in reforming processes and reducing costs. However, from the perspective of development, despite the wide variety of enzymes, the number of immobilized enzymes is relatively limited, and there are still only a few companies that use immobilized enzyme technology for large-scale production. Moreover, the immobilization of enzymes requires comprehensive consideration of many factors, such as enzyme activity, stability, optimal temperature and pH, suitable immobilization methods and carriers, etc. Therefore, the technology of immobilized enzymes still faces huge challenges and needs to be further developed and utilized.

[0006] With the development of my country's industry, the large-scale use of phenolic compounds has also brought new challenges to the protection of soil and water quality in the environment. How to effectively remove phenolic compounds from wastewater has become one of the important issues in the field of environmental protection. Enzymes, as efficient biocatalysts, can trigger specific reactions. Compared with other chemical catalysts, enzymes have ideal qualities such as high efficiency, high selectivity and the ability to operate under milder conditions. Their natural origin also makes them biodegradable and low in environmental impact, making them environmentally friendly. By optimizing the stability and activity of enzymes, it is expected to achieve efficient and rapid degradation of phenolic pollutants, contributing to environmental protection and sustainable development.

[0007] In summary, there are some challenges in the application of free Zanthoxylum bungeanum peroxidase in the removal of phenolic wastewater, such as susceptibility to harsh environments, poor stability, and difficulty in recycling and reuse, which seriously limits the practical application of Zanthoxylum bungeanum peroxidase. Summary of the invention

[0008] In view of the problems of the prior art, the present invention provides an immobilized Zanthoxylum bungeanum peroxidase enzyme and a preparation method and use thereof, which is used as a bioremediation agent for phenolic wastewater.

[0009] The invention provides a bamboo leaf prickly ash peroxidase immobilized enzyme, which comprises bamboo leaf prickly ash peroxidase and a magnetic carrier; the magnetic carrier is a magnetic material with phenylboric acid on the surface; the mass ratio of the phenylboric acid to the magnetic material is 6-8:1;

[0010] The mass ratio of the Zanthoxylum bungeanum peroxidase to the magnetic carrier is 1:15-30.

[0011] Preferably, the magnetic carrier is a magnetic agarose microsphere with surface modified with phenylboronic acid.

[0012] Preferably, the magnetic agarose microspheres with surface modification of phenylboronic acid are purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., with the serial number P65-010.

[0013] Preferably, the method for fixing the Zanthoxylum bungeanum peroxidase on a magnetic carrier is: reacting the magnetic carrier with the Zanthoxylum bungeanum peroxidase to obtain the product.

[0014] Preferably, in the reaction, the mass concentration of Zanthoxylum bungeanum peroxidase is 1-2 mg / mL.

[0015] Preferably, the rotation speed of the reaction is 90-120 rpm; and / or the temperature of the reaction is 20-30°C.

[0016] Preferably, the pH of the reaction is 7.0-8.0.

[0017] Preferably, the reaction time is 60 to 120 minutes.

[0018] The present invention provides a method for preparing any of the above-mentioned immobilized enzymes, which comprises reacting a magnetic carrier with Zanthoxylum bungeanum peroxidase to obtain the immobilized enzyme.

[0019] The present invention also provides the use of any of the above-mentioned immobilized enzymes in removing phenol and / or in a bioremediation agent for phenolic wastewater.

[0020] The invention has the beneficial effects of enhancing the pH, temperature and metal ion stability of the Zanthoxylum bungeanum peroxidase, and improving the circulation capacity and storage stability of the enzyme. When applied to the degradation of phenol, the invention has better temperature stability, pH stability and recyclability, and has a wider temperature and pH adaptability range. The phenol degradation rate is much higher than that of ZADTP in the range of pH 6.0-10.0. Moreover, the phenol solution treated with the immobilized enzyme has significantly reduced toxicity to normal human cells, thereby promoting the development of the Zanthoxylum bungeanum peroxidase in the degradation of phenolic wastewater, and has good industrial application prospects.

[0021] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0022] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation process of Zanthoxylum bungeanum peroxidase immobilized enzyme (ZADTP@MAM@PA).

[0024] Figure 2 Figure 1 shows the morphological characteristics of ZADTP@MAM@PA. A is an electron microscope scan of the magnetic carrier MAM@PA, B is an energy dispersive X-ray spectrum (EDS) of the immobilized enzyme ZADTP@MAM@PA, and C is an electron microscope scan of the immobilized enzyme. D is an elemental composition diagram of the immobilized enzyme ZADTP@MAM@PA.

[0025] Figure 3 Fourier transform infrared spectra of magnetic carrier MAM@PA and immobilized enzyme ZADTP@MAM@PA.

[0026] Figure 4X-ray diffraction (XRD) patterns of the magnetic carrier MAM@PA and the immobilized enzyme ZADTP@MAM@PA.

[0027] Figure 5 Hysteresis loop diagram of magnetic carrier MAM@PA and immobilized enzyme ZADTP@MAM@PA.

[0028] Figure 6 This is the result diagram of the effect of pH on enzyme immobilization.

[0029] Figure 7 This is the result graph showing the effect of ZADTP concentration on enzyme immobilization.

[0030] Figure 8 This is the effect of reaction time on enzyme immobilization.

[0031] Fig. 9 This is the result diagram of the effect of reaction temperature on enzyme immobilization.

[0032] Fig.10 The figure shows the immobilized enzyme loading capacity on different magnetic carriers.

[0033] Fig.11 The figure shows the enzyme activity retention rate of immobilized enzymes on different magnetic carriers.

[0034] Fig.12 Optimal pH and pH stability diagram of ZADTP and ZADTP@MAM@PA.

[0035] Fig.13 Optimum temperature and temperature stability diagram of ZADTP and ZADTP@MAM@PA.

[0036] Fig.14 Lin-Bayesian graphs of ZADTP and ZADTP@MAM@PA.

[0037] Fig.15 Figure 2 is the recycling capability diagram of ZADTP@MAM@PA.

[0038] Fig.16 This figure shows the effect of storage time on the enzyme activity of ZADTP and ZADTP@MAM@PA.

[0039] Fig.17 This is a graph showing the effect of different materials on the degradation rate of phenol.

[0040] Fig.18 This figure shows the effect of immobilized enzymes on different magnetic carriers on the phenol clearance rate.

[0041] Fig.19 This is a graph showing the effect of different temperatures on the degradation of phenol by ZADTP@MAM@PA.

[0042] Fig. 20 This is a graph showing the effect of different temperatures on the degradation of phenol by ZADTP.

[0043] Fig.21 This figure shows the effect of different pH on the degradation of phenol by ZADTP and ZADTP@MAM@PA.

[0044] Fig. 22 This is the diagram of the number of times ZADTP@MAM@PA can be reused to degrade phenol.

[0045] Fig.23 This is a graph showing the effect of phenol solution treatment on the survival rate of LO2 cells. DETAILED DESCRIPTION

[0046] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0047] Magnetic carrier agarose phenylboronic acid microspheres (MAM@PA): Suzhou Yingruicheng Biochemical Technology Co., Ltd., product number P65-010, batch number: A20230726-1.

[0048] Example 1 Immobilized enzyme of Zanthoxylum bungeanum peroxidase and its preparation method

[0049] This embodiment provides a bamboo leaf prickly ash peroxidase immobilized enzyme, and its preparation method is as follows Figure 1 As shown, the specific steps include:

[0050] (1) Extraction and purification of Zanthoxylum bungeanum peroxidase:

[0051] Take 100g fresh bamboo leaf pepper sample, add 500mL 0.05mM phosphate buffer solution (containing 2% polyvinyl pyrrolidone, pH 6.5), homogenize, and filter the mixture with four layers of gauze after standing at 4°C for 10h to obtain a crude enzyme extract. Saturate the crude enzyme solution with 15% (w / v) ammonium sulfate, then centrifuge (4000r / min, 10min) to remove the lower precipitate. Collect the supernatant, add 45% (w / v) ammonium sulfate to saturate again, then add tert-butanol to the sample at a solution volume ratio of 1:1, incubate at 25°C for one hour, and centrifuge again to promote phase separation. Collect the intermediate layer precipitate, dialyze for desalting, and freeze-dry to obtain ZADTP preliminarily purified after three-phase extraction. The freeze-dried ZADTP enzyme solution is loaded with 5mL of 50mM, pH6.5 phosphate solution. The SuperTandex-75 gel filtration column was equilibrated with 50 mM, pH 6.5 phosphate buffer, and the enzyme was eluted from the column at 0.4 mL / min, and 4 mL fractions were collected. The eluted fractions were monitored at 280 nm, and the enzyme activity was measured at 470 nm. The fractions with peroxidase activity from SuperTandex-75 were combined, dialyzed to remove salts, freeze-dried, and stored at -20 ° C until use.

[0052] (2) Preparation of immobilized Zanthoxylum bungeanum peroxidase enzyme ZADTP@MAM@PA:

[0053] Weigh 6.0 mg MAM@PA, add 200 μL of 1 mg / mL ZADTP enzyme solution (0.1 M PBS, pH = 8.0), and mix and culture the mixed solution at 120 rpm, 25 ° C for 90 min. Collect the immobilized enzyme material by magnetic separation and wash with phosphate buffer to remove non-specific unbound enzyme. Finally, the immobilized enzyme was redispersed in buffer (200 μL) and stored at 4 ° C.

[0054] The scanning electron microscopy results, infrared spectrum analysis, X-ray diffraction analysis and hysteresis loop results of ZADTP@MAM@PA prepared in this example are shown in Figure 2-5 shown.

[0055] The technical solution of the present invention is further described below through experiments. The immobilized enzymes used in the following experimental examples are all prepared according to the method of Example 1.

[0056] Experimental Example 1 Screening of preparation conditions for immobilized enzyme ZADTP@MAM@PA

[0057] 1. Experimental Methods

[0058] 1. Determination method of solid loading

[0059] The protein content was determined using the Bradford assay.

[0060] (1) Solution preparation: Weigh 50.0 mg of Coomassie Brilliant Blue G-250 and dissolve it completely in 25.0 mL of 95% ethanol solution. Mix well and then slowly add 50.0 mL of 85% (m / v) phosphoric acid. Mix well and dilute to 500 mL with distilled water at pH 7.0 to obtain Bradford reagent. Store at 4°C.

[0061] (2) Preparation of standard curve: Using bovine serum albumin (5 mg / mL) as the standard protein, prepare 100 μL of each of the standard protein solutions of 0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, add 5.0 mL of Coomassie Brilliant Blue solution to each tube, mix well, react at 25°C for 5 min, and measure the absorbance at 595 nm. Plot the standard curve with absorbance as the ordinate and protein concentration (μg / mL) as the abscissa: Y1=0.7363X1+0.0066, R=0.9998 (Y1: absorbance, X1: bovine serum albumin content in mg / mL).

[0062] (3) Determination of enzyme protein content in supernatant: Take 100 μL of ZADTP supernatant enzyme solution and washing solution, add 5 mL of Coomassie Brilliant Blue solution to each, react at 25°C for 5 min, measure the absorbance at 595 nm, and calculate the enzyme content in the supernatant and washing solution according to the standard curve. The enzyme loading amount is calculated as follows:

[0063]

[0064] 2. Enzyme activity determination method

[0065] Free enzyme activity determination method: The peroxidase activity is determined by colorimetry, with guaiacol as the substrate. At 470nm, peroxidase catalyzes the reaction of hydrogen peroxide and substrate to generate colored compounds, resulting in an increase in absorbance. Take 0.5mL 50mmol / L guaiacol and 0.5mL 50mmol / L H2O2 in a test tube, then add 4mL buffer solution (pH=6.5, 0.05mM phosphate buffer), mix well, add 500μL enzyme solution to start the reaction, quickly mix well, quickly pour into a colorimetric dish, and measure the rate of increase of the absorbance value at 470nm. One enzyme activity unit (U) is defined as the amount of enzyme required for the absorbance to change by 0.01 per minute, and repeat the measurement 3 times. The specific enzyme activity and relative enzyme activity of peroxidase are calculated using the following formulas:

[0066]

[0067] Method for determining the activity of immobilized enzyme: The activity of peroxidase is determined by colorimetry, with guaiacol as the substrate. At 470nm, the immobilized enzyme catalyzes the reaction of hydrogen peroxide and substrate to generate colored compounds, resulting in an increase in absorbance. Take 1mL of 25mmol / L guaiacol and 1mL of 25mmol / L H2O2 in a test tube, add 3mL of buffer solution (pH=6.5, 0.05mM phosphate buffer), mix well, add 5mg of immobilized enzyme, mix well for 2min, pour into a colorimetric dish, and measure the rate of increase of the absorbance value at 470nm. One enzyme activity unit (U) is defined as the amount of enzyme required for the absorbance to change by 0.01 per minute, and repeat the measurement 3 times. The enzyme activity retention rate of the immobilized enzyme is calculated using the following formula:

[0068]

[0069] 3. Single factor study of ZADTP@MAM@PA immobilization

[0070] The boron hydroxyl group on phenylboronic acid in magnetic agarose phenylboronic acid microspheres was covalently bonded with the cis-ortho-dihydroxyl group on the glycoprotein to form a stable five-membered ring structure, and the immobilized enzyme ZADTP@MAM@PA was obtained. Through single-factor experiments, the effects of buffer pH, Zanthoxylum bungeanum peroxidase concentration, immobilization time, and immobilization temperature on the enzyme immobilization effect were explored with the immobilization amount as an indicator, and the optimal preparation process of Zanthoxylum bungeanum peroxidase immobilized on magnetic agarose phenylboronic acid microspheres (MAM@PA) was obtained.

[0071] 4. Preparation of immobilized enzyme at different pH

[0072] The method for preparing ZADTP@MAM@PA was followed as in step (2) of Example 1, except that the pH of the ZADTP enzyme solution was 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively, and the mixed culture was performed for 60 min, and the immobilized enzyme obtained was finally measured for its immobilized capacity and enzyme activity retention rate.

[0073] 4. Preparation of immobilized enzyme at different ZADTP concentrations

[0074] The method for preparing ZADTP@MAM@PA was followed as in step (2) of Example 1, except that the concentrations of the ZADTP enzyme solutions were 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, and 1.6 mg / mL, respectively, and the solutions were mixed and cultured for 60 min, and the immobilized enzymes were finally obtained and their immobilized loading and enzyme activity retention rates were measured.

[0075] 5. Preparation of immobilized enzymes at different reaction times

[0076] The method for preparing ZADTP@MAM@PA was followed as in step (2) of Example 1, except that the mixed culture time was 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. The immobilized enzyme obtained was tested for its immobilized capacity and enzyme activity retention rate.

[0077] 6. Preparation of immobilized enzymes at different reaction temperatures

[0078] The method for preparing ZADTP@MAM@PA was carried out according to step (2) of Example 1, except that the mixed culture was carried out at temperatures of 20°C, 25°C, 30°C, 35°C and 40°C, respectively. The immobilized enzyme obtained was then tested for its immobilized capacity and enzyme activity retention rate.

[0079] 2. Experimental Results

[0080] 1. Effect of pH on enzyme immobilization

[0081] Changes in pH can affect the dissociation state of the protein surface charge, thereby affecting the composite behavior of Zanthoxylum peroxidase and MAM@PA materials. Therefore, the pH value of the reaction system is a key factor to consider during enzyme immobilization. Figure 6 As shown in the figure: the enzyme activity retention rate and the solid loading amount of the immobilized enzyme both showed a trend of first increasing and then decreasing with the increase of pH value. At a pH value of 8.0, the enzyme activity retention rate and the solid loading amount reached the highest values, 78.39% and 25.88 mg / g, respectively. The results show that an overly acidic or alkaline environment will affect the activity of the enzyme and the binding of the enzyme to the MAM@PA carrier, and ultimately affect the immobilization effect. When the pH is 8.0, the enzyme immobilization effect is the best.

[0082] 2. Effect of ZADTP concentration on enzyme immobilization

[0083] The results are as follows Figure 7 As shown: When the enzyme solution concentration is below 1 mg / mL, the immobilized capacity increases with the increase of enzyme solution concentration. When the enzyme solution concentration reaches 1 mg / mL, the immobilized capacity and enzyme activity retention rate reach the highest values, which are 26.21 mg / g and 70.20%, respectively. Continuing to increase the enzyme solution concentration, the immobilized capacity decreases slightly. The results show that when the ZADTP concentration is 1.0 mg / mL, the enzyme immobilization effect is the best.

[0084] 3. Effect of mixed culture time on enzyme immobilization

[0085] The results are as follows Figure 8As shown in the figure, the immobilization amount and relative enzyme activity retention rate increase with the increase of mixed culture time. At 90 minutes, the immobilization amount and enzyme activity retention rate reach the maximum, which are 38.94 mg / g and 79.99%, respectively. However, if the time is extended, there is almost no significant change in both the immobilization amount and the enzyme activity retention rate. The results show that when ZADTP is fixed to the MAM@PA carrier, the optimal mixed culture time is 90 minutes.

[0086] 4. Effect of reaction system temperature on enzyme immobilization

[0087] The results are as follows Fig. 9 As shown: When the temperature is less than 30°C, the solid loading capacity is greater than 35 mg / g, and the enzyme activity retention rate is higher than 75%. At 25°C, the solid loading capacity and enzyme activity retention rate both reach the highest values, 38.50 mg / g and 80.21%, respectively. When the temperature exceeds 30°C, the solid loading capacity and enzyme activity retention rate decrease rapidly. The results show that when ZADTP is fixed to the MAM@PA carrier, the temperature of the system is best controlled at 25°C.

[0088] The above results show that the best fixation effect can be achieved by fixing ZADTP to the MAM@PA carrier under the conditions of pH 8.0, temperature 25°C, ZADTP concentration 1.0 mg / g, and mixed culture time 90 min.

[0089] Experimental Example 2 Study on the enzymatic properties of immobilized enzyme ZADTP@MAM@PA

[0090] 1. Experimental Methods

[0091] 1. Preparation of immobilized Zanthoxylum bungeanum peroxidase on different magnetic carriers

[0092] (1) Preparation of immobilized Zanthoxylum bungeanum peroxidase (ZADTP@Fe3O4@GO) based on magnetic graphene oxide

[0093] (a) Extraction and purification of Zanthoxylum bungeanum peroxidase

[0094] Follow the method of step (1) of Example 1.

[0095] (b) Preparation of magnetic graphene oxide (Fe3O4@GO) carrier

[0096] Disperse 400 mg of graphene in 50 mL of concentrated nitric acid and stir magnetically at 60 ° C for 8 hours to obtain graphene oxide. Wash the graphene oxide six times with deionized water and dry it in a vacuum at 50 ° C. Take 150 mg of graphene oxide and 200 mg of ferric chloride and disperse them in 40 mL of ethylene glycol (containing 0.15 g of trisodium citrate, 1.8 g of sodium acetate, and 1.0 g of polyethylene glycol-20000) and stir magnetically for 2 hours. Seal the mixture in a reactor and heat it at 200 ° C for 12 hours. Finally, wash the obtained magnetic graphene oxide with deionized water and ethanol, and separate and collect it with a magnet to obtain magnetic Fe3O4@GO.

[0097] (c) Preparation of ZADTP@Fe3O4@GO

[0098] Weigh 6.0 mg of magnetic graphene oxide material / 3 μm-PS@Mag@COOH magnetic polymer beads and disperse them into 900 μL of 1.0 mg / mL ZADTP enzyme solution, followed by 100 μL of 25 mg / ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 μL of 25 mg / mL N-hydroxysuccinimide. The mixture was incubated at 150 rpm and 25 ° C for 8 hours. The immobilized enzyme material was collected by magnetic separation and washed with buffer to remove non-specific unbound enzymes. The immobilized enzyme was then redispersed in buffer (1 mL) and stored at 4 ° C.

[0099] (2) Preparation of immobilized Zanthoxylum bungeanum peroxidase (ZADTP@3μm-PS@Mag@COOH) using magnetic polymer beads as carriers

[0100] (a) Extraction and purification of Zanthoxylum bungeanum peroxidase

[0101] Follow the method of step (1) of Example 1.

[0102] (b) Preparation of magnetic polymer beads (3 μm-PS@Mag@COOH) carrier

[0103] Dispersion polymerization was carried out in a 500 mL glass kettle equipped with a nitrogen port, a stirrer and a Teflon gasket. 3.0 g of polyvinyl pyrrolidone K30 (molecular weight = 40,000) was dissolved in a mixed solvent of 120 mL of ethanol and 20 mL of water. Then, 0.28 g of azobisisobutyronitrile, 30 mL of styrene, 1.5 g of ethylene glycol dimethacrylate and 1.6 g of glycidyl methacrylate were added. The mixture was stirred for 30 minutes under nitrogen protection until a uniform solution was formed. The glass kettle was placed in a 75 ° C water bath and heated, and the reaction was continued for 12 hours and then cooled. The polystyrene microspheres modified with epoxy resin were separated by centrifugation of the reaction mixture, the supernatant was discarded and the microspheres were redispersed in ethanol. The microspheres were then dispersed in a mixed solvent of 10 mL of water and 60 mL of ethanol, and 3.0 g of ethylenediamine was added. The obtained microspheres (3 μm ammoniated polystyrene microspheres) were washed three times with deionized water and ethanol in sequence, and then dried under vacuum at 60° C. overnight.

[0104] Take 500 mg of dried microspheres and add them to 30 mL of deionized water, and disperse them ultrasonically for 20 minutes. Then add 10 mL of iron salt solution containing 0.5 g of ferrous sulfate and 1.0 g of ferric chloride. The mixture is mechanically stirred for 120 minutes under nitrogen protection, and then 10 mL of 15% ammonia water is added. The obtained dispersion system is heated at 75°C for 60 minutes. The prepared microspheres (named PS@Mag) are washed with deionized water and ethanol in turn, and then dried overnight at 60°C under vacuum.

[0105] 500 mg of dried PS@Mag microspheres were dispersed in 100 ml of mixed solvent (mixed with dimethyl sulfoxide and water in a volume ratio of 3:7, and 0.1 M sodium bicarbonate was added to adjust the pH to 10.5). Then 1.0 g of glycidyl methacrylate was added. After reacting at 50 ° C for 12 hours, the obtained microspheres were washed three times with ethanol and then dissolved in 50 mL of dimethyl sulfoxide. 5.0 mL of monomer (composed of divinylbenzene and methacrylic acid in a volume ratio of 1:8) and 0.2 g of azobisisobutyronitrile (initiator) were added. Nitrogen was introduced, and then polymerization was carried out at 75 ° C for 12 hours. The final product (named 3μm-PS@Mag@COOH) was washed three times with deionized water and ethanol respectively, and dried at room temperature.

[0106] (c) Preparation of ZADTP@3μm-PS@Mag@COOH

[0107] The method of step (c) of experimental method (1) of experimental example 2 was followed, except that the magnetic graphene oxide was replaced by 3 μm-PS@Mag@COOH magnetic polymer beads.

[0108] 2. Determination of enzyme activity of enzymes immobilized on different carriers

[0109] The immobilized enzymes ZADTP@MAM@PA, ZADTP@Fe3O4@GO and ZADTP@3μm-PS@Mag@COOH on three different magnetic carriers were tested for immobilization rate and enzyme activity according to the methods 1-2 of Experimental Example 1.

[0110] 3. Determination of ZADTP@MAM@PA enzyme activity at different pH

[0111] The enzyme activity was determined according to the method of Experimental Example 1 Experimental Method 2, except that the ZADTP enzyme freeze-dried powder and ZADTP@MAM@PA were dispersed in buffer solutions with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and the reaction temperature was 25°C. Samples were taken at 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, and 240 min to determine the enzyme activity, and the highest enzyme activity was set to 100%. The relative enzyme activity of ZADTP and ZADTP@MAM@PA at each pH was calculated to determine the optimal pH. The untreated enzyme solution was used as a control, and the relative enzyme activity was 100%. The relative enzyme activity of ZADTP and ZADTP@MAM@PA at each pH was calculated to investigate pH stability.

[0112] The buffer solution of pH 3.0 uses glycine hydrochloride buffer solution, the buffer solution of pH 4.0-5.0 uses acetate buffer solution, the buffer solution of pH 6.0-8.0 uses phosphate buffer solution, and the buffer solution of pH 9.0 uses tris(hydroxymethyl)aminomethane hydrochloride buffer solution.

[0113] 4. Determination of ZADTP@MAM@PA enzyme activity at different temperatures

[0114] The enzyme activities were measured according to the method of experimental method 2 of experimental example 1, except that the enzyme solution was kept warm for 30 minutes at 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C, and then samples were taken at 40 minutes, 80 minutes, 120 minutes, 160 minutes, 200 minutes, and 240 minutes to measure the enzyme activities of ZADTP and ZADTP@MAM@PA. The highest enzyme activity was set to 100%, and the relative enzyme activities of ZADTP and ZADTP@MAM@PA at each temperature were calculated to determine the optimal temperature. The untreated enzyme solution was used as a control, and the relative enzyme activity was 100%. The relative enzyme activities of ZADTP and ZADTP@MAM@PA at each temperature were calculated to investigate the temperature stability.

[0115] 5. Determination of Michaelis constant

[0116] The enzyme activities were determined according to the method of Experimental Example 1 Experimental Method 2, except that the H2O2 concentration was 20 mmol / L, and the enzyme activities were determined in the range of 1 mmol / L-30 mmol / L guaiacol concentration. The reciprocal of the absorbance change per unit time was used as the ordinate, and the reciprocal of the substrate concentration was used as the abscissa. The Lineweaver-Burk double reciprocal plotting method was used to obtain two straight lines. The Michaelis constant K of the free Zanthoxylum peroxidase and the immobilized enzyme under this condition was calculated according to the following formula: m and the maximum reaction rate V max value.

[0117]

[0118] Where is the change in absorbance per unit time, [S] is the concentration of the substrate guaiacol, V max Response rate, K m is the Michaelis constant.

[0119] 6. Effects of different metal ions on the enzyme activity of ZADTP@MAM@PA

[0120] The enzyme activities were measured according to the method of Experimental Example 1 Experimental Method 2, except that NaCl, K2SO4, CaCl2, MgSO4, ZnSO4, CuSO4 and MnSO4 were mixed with the ZADTP@MAM@PA solution and then added to the reaction solution after being left for 1 hour, so that the concentration of the compound in the reaction solution was 5 mM. The enzyme activities were then measured. The enzyme activities measured without metal ions or inhibitors were used as blank controls.

[0121] 7. Determination of the cyclic capacity of ZADTP@MAM@PA

[0122] The enzyme activity was determined according to the method of Experimental Example 1 Experimental Method 2, except that the free enzyme activity was not determined, but the enzyme activity was determined by ZADTP@MAM@PA. After the reaction, the immobilized enzyme ZADTP@MAM@PA was separated with a permanent magnet, and the separated enzyme was washed twice with a phosphate buffer solution, and then used in a new round of experiments and enzyme activity was determined. This cycle was repeated many times, and the enzyme activity of the immobilized enzyme measured for the first time was used as a control, set to 100%, and the relative enzyme activity of ZADTP@MAM@PA after n cycles was calculated.

[0123] 8. Storage stability determination of ZADTP@MAM@PA

[0124] Prepare free enzyme solution and immobilized enzyme ZADTP@MAM@PA solution of the same concentration, store at 4°C, pH 6.0, and measure enzyme activity according to the method of Experimental Example 1 Experimental Method 2. Measure enzyme activity changes every 5 days for 50 consecutive days, and equilibrate the enzyme solution to room temperature before each measurement. Measure the initial enzyme activity of the two enzyme solutions before the storage experiment, set it as 100%, and calculate the relative enzyme activity at different days.

[0125] 2. Experimental Results

[0126] 1. Study on the effect of carrier on the enzyme activity of immobilized enzyme

[0127] The results of solid loading are as follows Fig.10 As shown: After 2h of fixation, the solid loading of MAM@PA microspheres, 3μm-PS@Mag@COOH and Fe3O4@GO were 38.41mg / g, 40.29mg / g and 36.44mg / g respectively. Fig.11 As shown in the figure: the enzyme activity retention of ZADTP@MAM@PA is 86.95%, while that of ZADTP@3μm-PS@Mag@COOH and ZADTP@Fe3O4@GO is 36.70% and 30.66%, respectively. The enzyme activity retention of ZADTP@MAM@PA is much higher than that of ZADTP@3μm-PS@Mag@COOH and ZADTP@Fe3O4@GO, while the free enzyme immobilization capacity is not much different. The results show that the selection of immobilized enzyme carrier is very important, and MAM@PA is the preferred immobilized enzyme carrier.

[0128] 2. Study on the optimal pH and pH stability of ZADTP@MAM@PA

[0129] The results are as follows Fig.12 As shown in the figure: When pH is equal to 6, the enzyme activity of the free enzyme ZADTP is the highest, and the enzyme activity of the immobilized enzyme ZADTP@MAM@PA is the highest when pH is equal to 7. In the pH range of 4.0-8.0, the immobilized enzyme exhibits higher enzyme activity than the free enzyme. After being placed in a buffer solution of pH 5.0-8.0 for 4 hours, the relative enzyme activity of ZADTP remains above 74.9%, and the relative enzyme activity of ZADTP@MAM@PA remains above 80%. In a buffer solution of pH 3.0-4.0, the relative enzyme activity of the free enzyme ZADTP is between 15% and 31%, and the relative enzyme activity of ZADTP@MAM@PA is between 23.7% and 34%. The results show that the immobilized enzyme ZADTP@MAM@PA has a wider pH adaptability range than the free enzyme ZADTP and has better pH stability.

[0130] 3. Study on the optimum temperature and temperature stability of ZADTP@MAM@PA

[0131] The results are as follows Fig.13 As shown: ZADTP exhibits the best enzyme activity at 50°C, and ZADTP@MAM@PA has the highest enzyme activity at 30°C. After reacting at 20°C to 70°C for 4 hours, the relative enzyme activity of ZADTP@MAM@PA remains above 70%, and the relative enzyme activity of ZADTP remains above 48.7%. The results show that the immobilized enzyme ZADTP@MAM@PA has better temperature stability than the free enzyme ZADTP.

[0132] 4. Analysis of Michaelis constant of ZADTP@MAM@PA

[0133] Michaelis constant (V max ,K m ) is the characteristic constant of the enzyme, K m It indicates the substrate concentration when the enzyme-catalyzed reaction rate reaches half of the maximum reaction rate. Its size is only related to the properties of the enzyme. m The larger the value, the smaller the affinity between the enzyme and the substrate. max It indicates the maximum reaction rate of the enzyme under full saturation. Fig.14 As shown in the figure, two straight lines were obtained by Lineweaver-Burk double reciprocal plotting method, showing the K values ​​of ZADTP and ZADTP@MAM@PA for the substrate guaiacol. m The affinity of ZADTP@MAM@PA to guaiacol was weakened, which showed that the immobilization process of enzyme had little effect on the catalytic efficiency and affinity of enzyme.

[0134] 5. Effect of metal ions on ZADTP@MAM@PA enzyme activity

[0135] The effects of different metal ions on the enzyme activity of ZADTP and ZADTP@MAM@PA are shown in Table 1. ZADTP@MAM@PA has a strong tolerance to metal ions and its metal ion stability is improved. 2+ The difference was obvious, from the original relative inhibitor to the activator, and the relative enzyme activity increased from 78.47% to 152.82%. 2+ , Mn 2+ Mg 2+ 、Zn 2+ It has a certain activation effect on both ZADTP and ZADTP@MAM@PA, and the activation effect on ZADTP@MAM@PA is more obvious. + and K +It had no significant effect on the activity of ZADTP and had a weak promoting effect on ZADTP@MAM@PA. The results showed that the immobilization of the enzyme protected the enzyme activity from the harmful effects of these metal ions and improved the metal ion stability of the enzyme.

[0136] Table 1. Effects of metal ions on the enzyme activity of ZADTP@MAM@PA

[0137]

[0138] 6. Study on the recycling capacity of ZADTP@MAM@PA

[0139] Cycling capacity is the most important characteristic for evaluating the performance of immobilized enzymes. Fig.15 As shown in the figure: the free enzyme ZADTP can only be used once and cannot be recovered by centrifugation or other methods. The relative enzyme activity of the immobilized enzyme ZADTP@MAM@PA is 78% after 5 cycles and 39% after 8 cycles. This shows that the immobilized enzyme ZADTP@MAM@PA has good recycling performance.

[0140] 7. Study on storage stability of ZADTP@MAM@PA

[0141] The storage stability of immobilized enzyme is another important indicator for evaluating enzyme performance. Fig.16 As shown in the figure: After 15 days of storage, the enzyme activities of ZADTP and ZADTP@MAM@PA were maintained well, without significant activity loss. After 30 days of storage, the enzyme activity of ZADTP@MAM@PA was 81.2%, which was significantly higher than that of ZADTP (49.3%). After 50 days of storage, most of ZADTP was inactivated, while ZADTP@MAM@PA still maintained 68% of its enzyme activity. This shows that the magnetic carrier has a good protective effect on the enzyme, and ZADTP@MAM@PA has strong storage stability.

[0142] The above results show that the immobilized enzyme ZADTP@MAM@PA can enhance the pH, temperature and metal ion stability of ZADTP while maintaining the high activity of the enzyme, thereby improving the enzyme's circulation capacity and storage stability.

[0143] Experimental Example 3 Effect of ZADTP@MAM@PA on the degradation of phenol

[0144] 1. Experimental Methods

[0145] 1. Determination method of phenol degradation rate

[0146] The phenol concentration during the reaction was determined by colorimetry.

[0147] Solution preparation: Potassium ferrocyanide (81.28 mmol / L) and 4-aminotipyrine (20.1 mmol / L) were prepared with 0.25 mM sodium bicarbonate solution.

[0148] Preparation of standard curve: Take 6.00mmol / L phenol mother solution to prepare 0mmol / L, 0.60mmol / L, 1.20mmol / L, 1.80mmol / L, 2.40mmol / L, 3.00mmol / L, 3.60mmol / L standard solutions, 100μL each, add 4.85mL distilled water, mix well, then add 25μL potassium ferrocyanide and 25μL 4-aminotipyrine. Mix well, measure the absorbance at 510nm after 10 minutes. Draw the standard curve with phenol concentration (mmol / L) as the horizontal axis and absorbance value as the vertical axis: Y2

[0149] =0.1491X2+0.0342, R=0.9994 (Y2: absorbance, X2: phenol content mmol / L).

[0150] Determination of phenol content in supernatant: Take 100μL of ZADTP supernatant enzyme solution, add 4.85mL of water, mix well, then add 25μL of potassium ferrocyanide and 25μL of 4-aminotipyrine. Mix well, fully color, and measure the absorbance at 510nm after 10 minutes. Calculate the phenol content in the supernatant according to the standard curve. The calculation formula for the degradation rate of phenol is as follows:

[0151]

[0152] 2. Determination of the removal effect of different materials on phenol

[0153] The degradation or adsorption of phenol by the material under five different conditions, namely, O2&PEG, MAM@PA, ZADTP@MAM@PA, ZADTP@MAM@PA+H2O2, and ZADTP@MAM@PA+H2O2+PEG, was determined. The specific experimental operation is as follows:

[0154] Divide into five groups and add the following into 5 mL centrifuge tubes:

[0155] ①0.15mL of 40mmol / L phenol solution, 1.6mL of 25mmol / L H2O2 solution and 0.25mL of 1.2mmol / L PEG solution.

[0156] ②0.15mL of 40mmol / L phenol solution and 10mg MAM@PA.

[0157] ③0.15mL of 40mmol / L phenol solution and 10mg ZADTP@MAM@PA.

[0158] ④0.15mL of 40mmol / L phenol solution, 10mg ZADTP@MAM@PA and 1.6mL of 25mmol / L H2O2 solution.

[0159] ⑤0.15mL of 40mmol / L phenol solution, 10mg ZADTP@MAM@PA, 1.6mL of 25mmol / L H2O2 solution and 0.25mL of 1.2mmol / L PEG solution.

[0160] Deionized water was added to the above reaction tubes to make the total reaction volume 2 mL. Samples were taken at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively, the phenol concentration in each reaction tube was measured, and the phenol degradation rate was calculated.

[0161] 3. Determination of the removal effect of enzymes immobilized on different carriers on phenol

[0162] The total reaction volume was controlled to be 2 mL, the H2O2 concentration was 10 mmol / L, and the phenol concentration was 2 mmol / L. Immobilized enzymes of three carriers were used respectively, and the immobilized enzyme concentration was 5 mg / mL. The rest was supplemented with deionized water and added to the reaction tube. Samples were taken at 0 min, 20 min, 40 min, 60 min, and 80 min, respectively, to determine the phenol concentration and calculate the phenol degradation rate.

[0163] 4. Determination of the effect of temperature on phenol removal

[0164] The total reaction volume was controlled to be 2 mL, the H2O2 concentration was 10 mmol / L, the phenol concentration was 2 mmol / L, the concentrations of free ZADTP and ZADTP@MAM@PA were controlled to be 0.2 mg / mL and 5 mg / mL, respectively. The rest was supplemented with deionized water and added to the reaction tube. The reaction solution was kept at 20°C, 30°C, 40°C, 50°C and 60°C for 30 min, respectively. Then H2O2 was added to start the reaction. Samples were taken at 30 min, 60 min, 90 min, 120 min, 150 min and 180 min, the phenol concentration was determined and the phenol degradation rate was calculated.

[0165] 5. Determination of pH removal effect on phenol

[0166] The total reaction volume was controlled to be 2 mL, the phenol concentration was 2 mmol / L, and the H2O2 concentration was 10 mmol / L. The concentrations of ZADTP and ZADTP@MAM@PA were controlled to be 0.20 mg / L and 5 mg / mL, respectively. The rest was supplemented with deionized water and added to the reaction tube. The pH values ​​of the reaction solutions were adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, with 0.1 mol / L buffer solution. Samples were taken at 120 min to determine the phenol concentration and calculate the phenol degradation rate.

[0167] The 0.1 mol / L buffer solutions used to adjust the pH of the reaction solution were: glycine hydrochloride buffer solution for pH 3.0, acetate buffer solution for pH 4.0-5.0, phosphate buffer solution for pH 6.0-8.0, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution for pH 9.0.

[0168] 6. Reusability test of ZADTP@MAM@PA for phenol removal

[0169] The total reaction volume was controlled to be 2 mL, the H2O2 concentration was 10 mmol / L, the phenol concentration was 2 mmol / L, and the ZADTP@MAM@PA concentration was 5 mg / mL. The rest was supplemented with deionized water and added to the reaction tube. Samples were taken at 120 min to measure the phenol concentration and calculate the phenol degradation rate. After the reaction, the immobilized enzyme ZADTP@MAM@PA was separated with a permanent magnet, rinsed three times with 0.1 M, pH 8.0 PBS buffer, and then re-added to a new reaction system for catalytic reaction. The phenol degradation rate of each reaction was measured and the operation was repeated 8 times.

[0170] 7. Cytotoxicity experiment of ZADTP@MAM@PA for phenol removal

[0171] The MTT method was used to test the cytotoxicity of phenol water before and after treatment with ZADTP@MAM@PA immobilized enzyme. Human normal stem cell LO2 cells were selected, and the LO2 cell suspension was inoculated in a 96-well plate at a density of 10 / mL, with 100μL per well. After the cells adhered to the wall, a blank group, a control group, and a treatment group were set up, with 5 replicates in each group. The blank group only added sterile water without inoculating cells; the control group added RPMI-1640 culture medium and normal cells; the treatment group added RPMI-1640 culture medium containing a final concentration of 2mmol / L phenol water, 5mmol / L phenol water, 10mmol / L phenol water, and different concentrations of phenol treated with ZADTP@MAM@PA; the cells were cultured for 24h at 37°C and 5% CO2. The added samples were sterilized in advance by filtering with a microporous membrane with a pore size of 0.22μM. The absorbance of each group at 490nm was determined according to the method in the MTT kit, and the cell survival rate was calculated according to the following formula:

[0172] Cell survival rate (%) = (Α 490处理 -Α 490空白 ) / (Α 490对照 -Α 490空白 )×100%

[0173] 2. Experimental Results

[0174] 1. Effect of different materials on phenol removal

[0175] The results are as follows Fig.17 As shown: When only H2O2 and PEG were added, the phenol concentration did not change and the phenol removal rate was 0, indicating that H2O2 and PEG alone had no degradation effect. When only the magnetic carrier was added, the phenol removal rate was about 18% after 3 hours, indicating that MAM@PA itself can adsorb a small amount of phenol. The ZADTP@MAM@PA immobilized enzyme alone had almost no degradation effect on phenol (10%), while the phenol removal rate reached 54.45% after adding H2O2. This shows that H2O2 can activate the enzymatic reaction, and ZADTP@MAM@PA combined with H2O2 can be used to degrade phenol.

[0176] 2. Effect of enzymes immobilized on different carriers on the removal of phenol

[0177] The results are as follows Fig.18 As shown: ZADTP@MAM@PA showed the highest phenol degradation rate, with a degradation rate of 92% within 1 hour and a degradation rate of 98% within 80 minutes. The results are similar to the results of the determination of the enzyme activity retention rate, so MAM@PA as a carrier of Zanthoxylum bungeanum peroxidase is the preferred carrier for removing phenol in the present invention.

[0178] 3. Effect of temperature on phenol removal

[0179] The results are as follows Fig.19 , Fig. 20 As shown in the figure: After ZADTP@MAM@PA was treated at 20℃-40℃ for 3h, the phenol degradation rate was 70.11%-95.20%, among which the phenol degradation effect was best at 20℃. However, when the temperature was greater than 40℃, the phenol degradation rates of ZADTP@MAM@PA and ZADTP decreased, but compared with ZADTP, ZADTP@MAM@PA could keep the phenol degradation rate unchanged for a longer time, which shows that the ability of the enzyme to degrade phenol decreased when exposed to high temperature for a long time, but the stability of ZADTP@MAM@PA was better than that of ZADTP.

[0180] 4. Effect of pH on phenol removal

[0181] The results are as follows Fig.21 As shown in the figure, the phenol degradation rate of ZADTP@MAM@PA is much higher than that of ZADTP in the pH range of 6.0-10.0. The best phenol removal effect is achieved at pH 8.0, with a phenol degradation rate of 92.68%, while that of ZADTP is only 75.61%. The results show that compared with ZADTP, the immobilized enzyme ZADTP@MAM@PA has a wider pH adaptability range and better phenol removal effect in alkaline environment.

[0182] 5. Reusability analysis of ZADTP@MAMA@PA for phenol removal

[0183] The results are as follows Fig. 22 As shown in the figure, the phenol degradation rate of ZADTP@MAMA@PA decreases with the increase of operation times. After 5 cycles, the phenol degradation rate can still reach 79.64%. Then the phenol degradation rate decreases faster until the 8th cycle, when the degradation rate is about 55.64%. This shows that ZADTP@MAM@PA has good recyclability.

[0184] 6. ZADTP@MAMA@PA for cytotoxicity assessment after phenol removal

[0185] Treated industrial wastewater is often used to replenish water in rivers, lakes and other water bodies, thereby regulating water levels, improving the ecological environment, and providing safe water for local residents. Therefore, the present invention measured the cytotoxicity of phenol solutions of different concentrations before and after treatment with ZADTP@MAM@PA immobilized enzyme. The results are shown in Fig.23As shown: 2mM-10mM phenol solution significantly reduced the survival rate of LO2 cells. After treatment with ZADTP@MAM@PA, the original concentration of 2mM phenol solution had almost no effect on the cell survival rate (97.66%), the original concentration of 5mM phenol solution cell survival rate increased from 52.12% to 79.66%, and the 10mM phenol solution cell survival rate increased from 20.33% to 65.66%. The results further showed that the immobilized enzyme ZADTP@MAM@PA has a significant effect on the degradation of phenol, and the water samples treated with ZADTP@MAM@PA have weak toxicity to normal human cells.

[0186] The above results show that compared with free ZADTP, the immobilized enzyme ZADTP@MAM@PA has better temperature stability, pH stability and recyclability when degrading phenol, and has a wider temperature and pH adaptability range. The phenol degradation rate is much higher than that of ZADTP in the pH range of 6.0-10.0. In addition, the phenol solution treated with ZADTP@MAM@PA has reduced toxicity to LO2 cells, and the cell survival rate reaches 97.66%. It can be used to replenish water in rivers, lakes and other water bodies, thereby regulating water levels and improving the ecological environment.

[0187] Through the above embodiments and experimental examples, it can be seen that the present invention provides an immobilized Zanthoxylum bungeanum peroxidase enzyme, which is used as a bioremediation agent for phenolic wastewater, overcoming the shortcomings of poor stability and difficult recovery of the free enzyme of Zanthoxylum bungeanum, and has excellent performances of high stability, high activity and high circulability, thereby promoting the development of Zanthoxylum bungeanum peroxidase in degrading phenolic wastewater, and has a good industrial application prospect.

Claims

1. A Zanthoxylum bungeanum peroxidase immobilized enzyme, characterized in that: It comprises Zanthoxylum bungeanum peroxidase and a magnetic carrier; the magnetic carrier is a magnetic material with phenylboric acid on the surface; the mass ratio of phenylboric acid to magnetic material is 6-8:1; The mass ratio of the Zanthoxylum bungeanum peroxidase to the magnetic carrier is 1:15-30.

2. The immobilized enzyme according to claim 1, characterized in that: The magnetic carrier is a magnetic agarose microsphere with phenylboronic acid modified on the surface.

3. The immobilized enzyme according to claim 2, characterized in that The magnetic agarose microspheres with surface modified phenylboronic acid were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., with the serial number P65-010.

4. The immobilized enzyme according to claim 1, characterized in that The method for fixing the Zanthoxylum bungeanum peroxidase on the magnetic carrier is as follows: the magnetic carrier and the Zanthoxylum bungeanum peroxidase are reacted to obtain the product.

5. The immobilized enzyme according to claim 4, characterized in that: In the reaction, the mass concentration of Zanthoxylum bungeanum peroxidase is 1-2 mg / mL.

6. The immobilized enzyme according to claim 4, characterized in that: The rotation speed of the reaction is 90-120 rpm; and / or the temperature of the reaction is 20-30°C.

7. The immobilized enzyme according to claim 4, characterized in that: The pH of the reaction is 7.0-8.

0.

8. The immobilized enzyme according to claim 4, characterized in that: The reaction time is 60 to 120 minutes.

9. The method for preparing the immobilized enzyme according to any one of claims 1 to 8, characterized in that: The magnetic carrier is reacted with Zanthoxylum bungeanum peroxidase to obtain the product.

10. Use of the immobilized enzyme according to any one of claims 1 to 8 in removing phenol and / or in bioremediation of phenolic wastewater.

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